The gastrointestinal tract is the most densely colonized region of the human body (Savage, Ann. Rev, Microbiol. 31, 107 (1977); Tannock, Normal microflora (Chapman and Hall, London 1995)) and the accumulated evidence indicates that this collection of microbes has a powerful influence on the host in which it resides. Comparisons between germ free and conventional animals have shown that many biochemical, physiological and immunological functions are influenced by the presence of the diverse and metabolically active bacterial community residing in the gastrointestinal tract (Marteau and Rambaud, FEMS Microbiol. Rev. 12, 207 (1993); Norin et al., Appl. Environ. Microbiol. 74, 1850 (1991); Tannock, supra). Lactobacilli are important residents of the microflora (Ahrne et al., J. Appl. Microbiol. 85, 88 (1998); Kimura et al., Appl. Environ. Microbial. 63, 3394 (1997)), and have been the subject of intense and growing interest because of their possible role in the maintenance of gastrointestinal health (Bengmark, Gut 42, 2 (1998)). Of immense importance to lactobacilli functioning in this role is the ability to endure in the harsh conditions of the gastrointestinal tract, where the gastric pH frequently falls below 2.0 in healthy individuals (McLauchlan et al., Gut 30, 573 (1998)).
Changes in extracellular pH have been shown to influence the expression of a variety of genes from many different bacteria (reviewed in Olson, Mol. Microbiol. 8, 5 (1993)). In the presence of a low external pH (&lt;3.5), L. acidophilus is able to maintain cytoplasmic pH at values close to neutral (Kashket, FEMS Microbiol. Rev. 46, 233 (1987)). However, the mechanisms by which L. acidophilus responds and adapts to extremely acidic conditions remain poorly defined, For several organisms that inhabit the gastrointestinal tract, the F.sub.1 F.sub.0 -ATPase is an important element in the response and tolerance to low pH. In the fermentative bacterium, Enterococcus (En.) hirae, maintenance of cytoplasmic pH has been shown to occur via amplification of the proton translocating ATPase (Kobayashi et al., J. Bacteriol. 158, 1157 (1984); Kobayashi et al., J. Biol. Chem. 261, 627 (1986)). Similarly, a short exposure of Salmonella typhimurium to pH 6.0 induces the synthesis of the F.sub.1 F.sub.0 -ATPase (Foster and Hall, J. Bacteriol. 173, 5129 (1990); Foster and Hall, J. Bacteriol. 172, 771 (1990)). Nanen and Hutkins (J. Dairy Sci. 74, 747 (1991)) have demonstrated that the specific activity of H.sup.+ -ATPases from several lactic acid bacteria increases as the extracellular pH moves from neutral to 5.0. Alternatively, changes in environmental pH appear to have little influence on the expression of the atp operon, whose genes code for the various subunits of the H.sup.+ -ATPase, in Escherichia coli (Kasimoglu et al., J. Bacteriol. 178, 5563 (1996)). Likewise, expression of the atp operon of Bacillus subtilis appears to be constitutive (Santana et al., J. Bacteriol. 176, 6802 (1994)).
The identification of conditionally expressed genes provides a wealth of insight into the physiological consequences of and responses to a given stimulus. In the case of L. acidophilus, a significant challenge remains in understanding the intestinal roles and activities of this organism. An important element in this regard is the determination of which characteristics are important for the survival and success of this organism in the gastrointestinal tract. While differential display (Liang and Pardee, Science 257, 967 (1992); Welsh et al., Nucleic Acids Res. 20, 4965 (1992)) has been used extensively to identify conditionally expressed genes in eukaryotes, the application of this methodology in prokaryotes has not been explored to a comparatively significant extent (Abu Kwaik and Pederson, Mol. Microbiol. 21, 543 (1996); Fislage, Electrophoresis 19, 613 (1998); Fislage et al., Nucleic Acids Res. 25, 1830 (1997); Wong and McClelland, Proc. Natl. Acad. Sci. USA 91, 639 (1994); Zhang and Normark, Science 273, 1234 (1996)). Practical problems with the method are presented by: the relatively large proportion of structural RNA species in the total RNA; the low level of polyadenylation of mRNA (Sarkar, Ann. Rev. Biochem. 66, 173 (1997)), which prohibits the use of 3' dT anchored primers; and the structural instability and short half life of low abundance mRNA species of prokaryotes as compared to eukaryotes (Higgins et al., Curr. Opin. Genet. Dev. 2, 739 (1992)).